DESCRIPTION: The long-term goal of this research is to determine the mechanisms for sorting and storage of regulated secretory proteins in salivary glands. The answer to this question is important for our understanding of the regulation of protein secretion from salivary glands and, hence, the composition of saliva. Significant progress towards this goal was made in the first grant period. It was shown that calcium- and pH-mediated aggregation do not contribute significantly to the storage of secretory proteins in parotid secretory granules, suggesting that sorting in this gland differs from that of other endocrine and exocrine glands. Indeed, it was demonstrated that sulfated proteoglycans are necessary for efficient storage of secretory proteins in secretory granules of rat parotid acinar cells. The sulfated proteoglycan appears to buffer the internal pH of secretory granules. These results led to the hypothesis that sulfated proteoglycans act as buffering agents that regulate protein sorting and storage in parotid acinar cells. Specific Aim 1 will test if sulfated proteoglycans act as buffering agents in parotid secretory granules. Aim 2 will test if expression and) post-translational modification of the proteoglycan core proteins regulates the sorting and storage of other) parotid secretory proteins. These Aims will employ isoproterenol treatment to overexpress acidic and basic PRPs in rat parotid glands combined with in vitro tissue culture approaches. As an alternative, we will use transgenic mice that overexpress PRPs in salivary glands. In Aim 3, it will be tested if sulfated proteoglycans play a role in protein or membrane binding of PSP. Specific Aim 4 will test if sulfated proteoglycans or other acidic proteins are involved in sorting and storage of secretory proteins in the submandibular gland, a current target for gene therapy protocols. The proposed research will use cell biological and molecular methods in salivary cells and protein expression experiments in intact animals. The results of this research will provide insight into the unique sorting mechanisms that function in salivary glands. This will lead to a better understanding of the formation of saliva and provide the basis for efficient delivery of therapeutic proteins from salivary glands.